The Automated Quantification Pipeline presented below is written in R version 3.6.3 with the following package dependencies:
Bioconductor::EBImage
stringr
shiny
shinyFiles
shinyjs
shinythemes
tidyr
The user supplies the following criteria:
If necessary, files can be converted from .nd2 to .tif files using FIJI’s built in “Batch Convert” macro.
Tif files are read in and stored in an N x 2 matrix of the form
\[ \left[ \begin{matrix} \text{Image 1 Filename} & \text{Image 1 Filepath} \\ \vdots & \vdots \\ \text{Image N Filename} & \text{Image N Filepath} \end{matrix} \right] \]
where N = number of files. The rows (images) are processed in sequential order.
A local channel object stores information about the channels of the image using the user-supplied channel ordering. For each channel, two image matrices are stored:
Matrix of original pixel intensities of the channel, used for quantification
Matrix of normalized pixel intensities of the channel, used for image manipulation and rendering
All normalized channels are then converted to grayscale.